Use of CDK9 inhibitors to reduce cartilage degradation

ABSTRACT

The present invention relates to the use of cyclin-dependent kinase 9 (CDK9) inhibitors to reduce, inhibit and/or prevent cartilage degradation. CDK9 inhibitors can be used to reduce, inhibit and/or prevent cartilage degradation and loss of cartilage viability during allograft storage. CDK9 inhibitors can be used as a post-injury intervention treatment to reduce, inhibit and/or prevent the acute cellular responses that lead to future cartilage degradation and osteoarthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.14/351,878, filed on Apr. 15, 2014 and issued as U.S. Pat. No. 9,498,471on Nov. 22, 2016, which is a U.S. national phase under 35 U.S.C. § 371of Intl. Appl. No. PCT/US2012/061079, filed on Oct. 19, 2012, whichclaims the benefit of under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/549,741, filed on Oct. 20, 2011, which are herebyincorporated herein by reference in their entireties for all purposes.

FIELD

The present invention relates to the use of cyclin-dependent kinase 9(CDK9) inhibitors to reduce, inhibit and/or prevent cartilagedegradation. CDK9 inhibitors can be used to reduce, inhibit and/orprevent cartilage degradation and loss of cartilage viability duringallograft storage. CDK9 inhibitors can be used as a post-injuryintervention treatment to reduce, inhibit and/or prevent the acutecellular responses that lead to future cartilage degradation andosteoarthritis.

BACKGROUND

A host of pro-inflammatory and cellular stress induces inflammatoryresponse in chondrocytes, leading to upregulation of matrixmetalloproteinases (MMPs) and aggrecanases that degrade the cartilagematrix. Chronic deregulation of these catabolic pathways is suspected ofcausing osteoarthritis. Regardless of the sources of inflammation, thedownstream signals all converge on a common mechanism that activatestranscription of all primary response genes. This regulatory point iscontrolled by the transcription factor cyclin-dependent kinase 9 (CDK9)and its T-type cyclin partner. It was believed for many years that therate-limiting step in transcriptional activation is the recruitment oftranscription factors and RNA Polymerase II (Pol II) to gene promoters.However, recent studies on primary response genes have shown that intheir basal and unstimulated states, Pol II is already pre-assembled butis paused at the promoters (Hargreaves, et al., Cell 2009, 138:129-45;Zippo, et al., Cell 2009, 138:1122-36). The rapid activation of thesegenes is the result of signal-induced recruitment of CDK9 to thepromoters, where it phosphorylates Pol II. Phosphorylation by CDK9induces a conformational change that allows Pol II to enter possessiveelongation to efficiently transcribe full-length mRNAs (Zhou and Yik,MMBR 2006, 70(3): 646-659). Given that CDK9 controls a common mechanismof transcriptional activation of inducible genes, it is an effectivetarget for inhibiting the undesirable inflammatory responses fromdiverse cellular stress, such as sports-related injuries. The presentinvention is based, in part, on the discovery that pharmacological CDK9inhibitors, e.g., flavopiridol, and analogs and salts thereof, caneffectively suppress primary inflammatory genes in human articularchondrocytes in vitro. Effective suppression of inflammatory responsesallows for longer storage life for osteochondral explants used commonlyin cartilage repair, and also has therapeutic implications in preventingcartilage breakdown in post-traumatic osteoarthritis.

SUMMARY

In one aspect, the invention provides methods of reducing, preventing orinhibiting cartilage degradation and/or chondrocyte death in a subjectin need thereof. In some embodiments, the methods comprise administeringto the subject an effective amount of an inhibitor of cyclin-dependentkinase 9 (CDK9), thereby reducing, preventing or inhibiting cartilagedegradation and/or chondrocyte death in the subject.

In a further aspect, the invention provides methods of reducing,preventing, delaying or inhibiting the onset and/or progression ofpost-traumatic osteoarthritis in a subject in need thereof. In someembodiments, the methods comprise administering to the subject aneffective amount of an inhibitor of cyclin-dependent kinase 9 (CDK9),thereby reducing, preventing or inhibiting post-traumatic osteoarthritisin the subject.

In some embodiments, the subject has experienced a traumatic injury tocartilage tissue. In some embodiments, the subject has undergone orreceived joint surgery (which can inflict traumatic injury tocartilage). In some embodiments, the inhibitor of CDK9 is administeredwithin 10 days, e.g., within 9, 8, 7, 6, 5, 4, 3, 2, 1 days, e.g.,within 24, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours afterexperiencing traumatic injury. In some embodiments, the subject hasundergone surgery to repair damaged cartilage tissue. In someembodiments, the subject has received an osteochondral explant, e.g., acartilage allograft. In some embodiments, the inhibitor of CDK9 isadministered concurrently with or prior to surgery. In some embodiments,the inhibitor of CDK9 is administered within 10 days, e.g., within 9, 8,7, 6, 5, 4, 3, 2, 1 days, e.g., within 24, 20, 18, 16, 14, 12, 10, 9, 8,7, 6, 5, 4, 3, 2, 1 hours after surgery. In some embodiments, theinhibitor of CDK9 is administered over a course of 10 days, e.g., over9, 8, 7, 6, 5, 4, 3, 2, 1 days. In various embodiments, the inhibitor ofCDK9 is administered every 2 days, every day, or twice daily, asappropriate.

In some embodiments, the inhibitor of CDK9 is administered systemically.In some embodiments, the inhibitor of CDK9 is administered directly tothe lesion, e.g., to the site of injured cartilage tissue.

In some embodiments, the inhibitor of CDK9 is a small organic compound,e.g., flavopiridol, and analogs and salts thereof. In some embodiments,the inhibitor of CDK9 is an inhibitory nucleic acid. In varyingembodiments, the inhibitor of CDK9 is flavopiridol and is administeredintravenously.

In another aspect, the invention provides methods of reducing,preventing or inhibiting degradation of an osteochondral explant (e.g.,ex vivo cartilage tissue) and/or chondrocyte death during storage. Insome embodiments, the methods comprise storing the cartilage in asolution comprising an effective amount of an inhibitor ofcyclin-dependent kinase 9 (CDK9). In some embodiments, the osteochondralexplant is allograft cartilage. In some embodiments, the inhibitor ofCDK9 is a small organic compound, e.g., flavopiridol, and analogs andsalts thereof. In some embodiments, the osteochondral explant issubmerged in the solution comprising the inhibitor of CDK9. In someembodiments, the solution comprises flavopiridol, or an analog or saltthereof, at a concentration in the range of about 100 nM to about 1000nM, e.g., about 300 nM. The solution may contain additionalpharmaceutically acceptable excipients, described herein.

In a related aspect, the invention provides compositions comprising anosteochondral explant (e.g., ex vivo cartilage tissue) in a solutioncomprising an inhibitor of cyclin-dependent kinase 9 (CDK9). In someembodiments, the osteochondral explant is allograft cartilage. In someembodiments, the inhibitor of CDK9 is a small organic compound, e.g.,flavopiridol, and analogs and salts thereof. In some embodiments, theosteochondral explant is submerged in the solution comprising theinhibitor of CDK9. In some embodiments, the solution comprisesflavopiridol, or an analog or salt thereof, at a concentration in therange of about 100 nM to about 1000 nM, e.g., about 300 nM. In varyingembodiments, the solution is a physiologically isotonic solution. Invarying embodiments, the solution is an aqueous solution. The solutionmay contain additional pharmaceutically acceptable excipients, describedherein. In some embodiments, the composition is provided as a packagedkit.

Definitions

The term “cyclin-dependent kinase 9” or “CDK9” refers to a member of thecyclin-dependent protein kinase (CDK) family. CDK family members arehighly similar to the gene products of S. cerevisiae cdc28, and S. pombecdc2, and known as important cell cycle regulators. CDK9 was found to bea component of the multiprotein complex TAK/P-TEFb, which is anelongation factor for RNA polymerase II-directed transcription andfunctions by phosphorylating the C-terminal domain of the largestsubunit of RNA polymerase II. CDK9 forms a complex with and is regulatedby its regulatory subunit cyclin T or cyclin K. HIV-1 Tat protein wasfound to interact with this protein and cyclin T. Structurally, “CDK9”refers to nucleic acids and polypeptide polymorphic variants, alleles,mutants, and interspecies homologs that: (1) have an amino acid sequencethat has greater than about 90% amino acid sequence identity, forexample, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater aminoacid sequence identity, preferably over a region of at least about 25,50, 100, 200, 400, or more amino acids, or over the full-length, to anamino acid sequence encoded by a CDK9 nucleic acid (see, e.g., GenBankAccession No. NM_001261.3); (2) bind to antibodies, e.g., polyclonalantibodies, raised against an immunogen comprising an amino acidsequence of a CDK9 polypeptide (e.g., GenBank Accession No.NP_001252.1); or an amino acid sequence encoded by a CDK9 nucleic acid(e.g., CDK9 polynucleotides described herein), and conservativelymodified variants thereof; (3) specifically hybridize under stringenthybridization conditions to an anti-sense strand corresponding to anucleic acid sequence encoding a CDK9 protein, and conservativelymodified variants thereof; (4) have a nucleic acid sequence that hasgreater than about 90%, preferably greater than about 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide sequence identity,preferably over a region of at least about 25, 50, 100, 200, 500, 1000,2000 or more nucleotides, or over the full-length, to a CDK9 nucleicacid (e.g., CDK9 polynucleotides, as described herein, and CDK9polynucleotides that encode CDK9 polypeptides, as described herein).Based on the knowledge of CDK9 homologs, those of skill can readilydetermine residue positions that are more tolerant to substitution. Forexample, amino acid residues conserved amongst species are less tolerantof substitution or deletion. Similarly, amino acid residues that are notconserved amongst species are more tolerant of substitution or deletion,while retaining the function of the CDK9 protein.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, α-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine I, Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and

7) Serine (S), Threonine (T)

(see, e.g., Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” and variants thereof in thecontext of two or more polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a specified percentage of aminoacid residues or nucleotides that are the same (i.e., at least 60%identity, optionally at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity over a specified region (or the wholereference sequence when not specified)), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. The present invention providespolypeptides substantially identical to CDK9, as described herein.Optionally, the identity exists over a region that is at least about 50amino acids in length, or more preferably over a region that is 100 to500 or 1000 or more amino acids in length, or over the full-length ofthe sequence.

The terms “similarity,” or “percent similarity,” and variants thereof inthe context of two or more polypeptide sequences, refer to two or moresequences or subsequences that have a specified percentage of amino acidresidues that are either the same or similar as defined in the 8conservative amino acid substitutions defined above (i.e., 60%,optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%similar over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Sequences having less than 100%similarity but that have at least one of the specified percentages aresaid to be “substantially similar.” Optionally, this identity existsover a region that is at least about 50 amino acids in length, or morepreferably over a region that is at least about 100 to 500 or 1000 ormore amino acids in length, or over the full-length of the sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The term “comparison window”, and variants thereof, includes referenceto a segment of any one of the number of contiguous positions selectedfrom the group consisting of from 20 to 600, usually about 50 to about200, more usually about 100 to about 150 in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequences for comparison are well known in the art. Optimalalignment of sequences for comparison can also be conducted by the localhomology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981),by the homology alignment algorithm of Needle man and Wunsch J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson andLipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis.), Karlin and Altschul Proc.Natl. Acad. Sci. (U.S.A.) 87:2264-2268(1990), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Examples of an algorithm that is suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information (onthe internet at ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. StandardBLAST algorithm parameters have an expected threshold of 10 (accordingto the stochastic model of Karlin and Altschul (PNAS,87:2264-2268(1990)); a word size of 28; reward and penalty of 1/−2 (aratio of 0.5, or 1/−2, is used for sequences that are 95% conserved);and a linear GAP cost.

The term “effective amount” refers to an amount (here of an inhibitor ofCDK9) which provides either subjective relief of a symptom(s) or anobjectively identifiable improvement as noted by the clinician or otherqualified observer. Determination of an effective amount is well withinthe capability of those skilled in the art, especially in light of thedetailed disclosure provided herein. Generally, an efficacious oreffective amount of a combination of one or more polypeptides of thepresent invention is determined by first administering a low dose orsmall amount of a polypeptide or composition and then incrementallyincreasing the administered dose or dosages, adding a second or thirdmedication as needed, until a desired effect of is observed in thetreated subject with minimal or no toxic side effects. Applicablemethods for determining an appropriate dose and dosing schedule foradministration of a combination of the present invention are described,for example, in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Edition, 2006, supra; in a Physicians' Desk Reference(PDR), 64^(th) Edition, 2010; in Remington: The Science and Practice ofPharmacy, 21^(st) Ed., 2006, supra; and in Martindale: The Complete DrugReference, Sweetman, 2005, London: Pharmaceutical Press., and inMartindale, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996,Amer Pharmaceutical Assn, each of which are hereby incorporated hereinby reference.

The terms “treating” and “treatment” and variants thereof refer topromoting healing, delaying the onset of, retarding or reversing theprogress of, alleviating or preventing either the disease or conditionto which the term applies (e.g., cartilage degradation), or one or moresymptoms of such disease or condition. Treating and treatment encompassboth therapeutic and prophylactic treatment regimens.

The terms “subject,” “patient,” or “individual” interchangeably refer toany mammal, for example, humans and non-human primates, domestic mammals(e.g., canine, feline), agricultural mammals (e.g., bovine, equine,ovine, porcine) and laboratory mammals (e.g., mouse, rat, rabbit,hamster).

As used herein, “administering” refers to local and systemicadministration, e.g., including enteral, parenteral, pulmonary, andtopical/transdermal administration. Routes of administration forcompounds (e.g., inhibitors of CDK9, e.g., flavopiridol, and analogs andsalts thereof) that find use in the methods described herein include,e.g., oral (per os (P.O.)) administration, nasal or inhalationadministration, administration as a suppository, topical contact,transdermal delivery (e.g., via a transdermal patch), intrathecal (IT)administration, intravenous (“iv”) administration, intraperitoneal(“ip”) administration, intramuscular (“im”) administration,intralesional administration, or subcutaneous (“sc”) administration, orthe implantation of a slow-release device e.g., a mini-osmotic pump, adepot formulation, etc., to a subject. Administration can be by anyroute including parenteral and transmucosal (e.g., oral, nasal, vaginal,rectal, or transdermal). Parenteral administration includes, e.g.,intravenous, intramuscular, intra-arterial, intradermal, subcutaneous,intraperitoneal, intraventricular, ionophoretic and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The terms “systemic administration” and “systemically administered”refer to a method of administering a compound or composition to a mammalso that the compound or composition is delivered to sites in the body,including the targeted site of pharmaceutical action, via thecirculatory system. Systemic administration includes, but is not limitedto, oral, intranasal, rectal and parenteral (e.g., other than throughthe alimentary tract, such as intramuscular, intravenous,intra-arterial, transdermal and subcutaneous) administration.

The term “co-administering” or “concurrent administration”, when used,for example with respect to the compounds (e.g., inhibitors of CDK9,e.g., flavopiridol, and analogs and salts thereof) and/or analogsthereof and another active agent, refers to administration of thecompound and/or analogs and the active agent such that both cansimultaneously achieve a physiological effect. The two agents, however,need not be administered together. In certain embodiments,administration of one agent can precede administration of the other.Simultaneous physiological effect need not necessarily require presenceof both agents in the circulation at the same time. However, in certainembodiments, co-administering typically results in both agents beingsimultaneously present in the body (e.g., in the plasma) at asignificant fraction (e.g., 20% or greater, preferably 30% or 40% orgreater, more preferably 50% or 60% or greater, most preferably 70% or80% or 90% or greater) of their maximum serum concentration for anygiven dose. For example, in various embodiments, an inhibitor of CDK9 isco-administered with protein complexes or protein scaffolds comprisingone or more monomers of cartilage oligomeric matrix protein (COMP) boundto one or more growth factors, as described in co-owned and co-pendingInternational Appl. No. PCT/US2011/051610.

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s)/compound(s) at issue to the subject. Causing to beadministered can involve diagnosis and/or determination of anappropriate therapeutic or prophylactic regimen, and/or prescribingparticular agent(s)/compounds for a subject. Such prescribing caninclude, for example, drafting a prescription form, annotating a medicalrecord, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that flavopiridol suppresses LPS-induced inflammatoryresponse. First-passage human chondrocytes grown in monolayer culturewere given a strong inflammatory insult consisting of 10 ng/mllipopolysaccharides (LPS) for 5 hours. This induced transcriptionalactivation of the primary response genes IL-1β and iNOS. Pre-treatmentwith 300 nM flavopiridol strongly attenuated transcriptional activationof both primary response genes by LPS. The effect of CDK9 inhibition ontranscription of 3 hours after the addition of LPS was also tested.Inhibition of CDK9 three hours after the inflammatory insult similarlyattenuated the transcriptional activation of primary response genesIL-1β and iNOS, showing that the window for therapeutic intervention canbe at least 3 hours after a joint injury event, making it a practicaltherapeutic strategy.

FIG. 2 illustrates that flavopiridol inhibits IL-1β-induced expressionof matrix-degrading enzymes in chondrocytes.

FIG. 3 illustrates that inhibition of CDK9 by flavopiridol effectivelysuppresses the activation of a broad range of primary inflammatoryresponse genes by IL-1β. Primary human chondrocytes from 3 donors weretreated with IL-1β with or without cdk9 inhibitor (300 nM flavopiridol).Gene expression was analyzed using PCR-Array for NFκB targets (Qiagen)on a HT-7900 instrument. IL-1β strongly activated the primary responsegenes. Inhibition of cdk9 activity almost completely abolishes theeffects of IL-1β. For example, IL-1β induced expression of IL-6 by492-fold, but only 4.2-fold in the presence of cdk9 inhibitor,representing a 99.2% repression of IL-1β-dependent inflammatory responsegene transcription. On average, across 3 donors, cdk9 inhibitionrepressed IL-1β activity by >86%, with respect to 54 inflammatoryresponse genes. Importantly, housekeeping genes are unaffected by eitherIL-1β or cdk9 inhibitors.

FIGS. 4A-B illustrate that the CDK9 inhibitor flavopiridol is effectiveagainst different inflammatory stimuli. Primary human articularchondrocytes (n=3 donors) in monolayer culture were treated withdifferent inflammatory stimuli (10 ng/ml of either IL-1β, LPS, or TNFα)with or without 300 nM Flavopiridol for 5 hours. iNOS mRNA wasquantified by real-time PCR as a measure of inflammatory response. Theinduction of iNOS by each stimulus alone was arbitrarily set to 100%(first bar) and compared to the respective value obtained in sampleco-treated with each inflammatory stimulus and Flavopiridol.

FIG. 5 illustrates that flavopiridol effectively suppresses theinduction of a broad range of inflammatory mediators. Primary humanchondrocytes (n=3) in monolayer culture were treated with 10 ng/ml IL-1βwith or without 300 nM Flavopiridol for 5 hours. Gene expression wasanalyzed using real-time PCR-Array for NFκB targets (Qiagen) asdescribed in Methods and shown here as heat map (Green=minimumexpression, Red=maximum expression). Listed are 54 out of 60NFκB-targeted genes tested that were induced >2-fold by IL-1β, whichstrongly activated these genes (compare lanes 1 and 2). Flavopiridolalmost completely abolishes the effects of IL-1β (lane 3). Importantly,housekeeping genes are unaffected by either IL-1β or Flavopiridol.

FIG. 6 illustrates that CDK9 inhibition prevents IL-1β-induced MMPs andADAMTS4 expression. Primary chondrocytes (n=3) were treated with 10ng/ml IL-1β with or without Flavopiridol for 5 hours, and the mRNAexpression of cartilage degrading enzymes MMP-1, -3, -9, -13, andADAMTS4 (aggrecanase) was determined by real-time PCR as described inMethods.

FIGS. 7A-B illustrate that CDK9 inhibition prevents IL-1β induced GAGbreakdown in cartilage. Human cartilage explants (n=5) were treated with1 ng/ml IL-1β and the indicated concentrations of Flavopiridol for 6days (media change at day 3). GAG released into the medium was measuredby DMMB assays and normalized to the wet weight of the explants.Treatment with IL-1β alone caused cartilage degradation as indicated byincreased GAG release. In the presence of Flavopiridol, GAG releasereturned to baseline.

FIG. 8 illustrates CDK9 inhibition prevents IL-1β-induced Col2abreakdown in cartilage. Human cartilage explants (n=5) were treated with1 ng/ml IL-1β and the indicated concentrations of Flavopiridol for 6days (media change at day 3). Cleaved Col2a peptides released into themedium was measured by C2C ELISA and normalized to the wet weight of theexplants as described in the Methods. Treatment with IL-1β alone causedcartilage degradation as indicated by increased Col2a peptides. In thepresence of Flavopiridol, Col2a peptides release returned to baseline.

FIG. 9 illustrates that inhibition of CDK9 with flavopiridol stronglyreduced the number of dead or dying cells in the cartilage explantsafter simulated injury with IL-1β. Human cartilage explants were treatedwith IL-1β to simulate injury, with or without CDK9 inhibitor. Celldeath was measured with a live/dead stain.

FIG. 10 illustrates application of a whole joint injury model toinitiate post-traumatic OA in mice, in which a single rapid non-invasivemechanical load induces anterior cruciate ligament (ACL) rupture. Togenerate knee injuries, the mouse is anesthetized using isofluraneinhalation, and then the right leg of each mouse is subjected to tibialcompression loading.

FIGS. 11A-B illustrate a typical histology of an injured joint andcontralateral uninjured joint 8-weeks post-injury. Safranin-O/Fast Greenstained mouse knee sections 8-weeks post-injury. A-Uninjuredcontralateral knee, B-Injured knee. Note the loss of proteoglycans,damaged meniscus, and calcification of the meniscus after injury.

FIG. 12 illustrates quantitative analysis of bone volume in injured andcontrol joints by μCT. Note the rapid and substantial (44%) loss ofsubchondral bone volume is reproducibly seen in the first few days afterinjury. Error bars indicate standard deviation, with n=6 for each datapoint.

FIG. 13 illustrates Cdk9 Inhibition Treatment Window. Injury wassimulated with a strong inflammatory stimulus (10 ng/ml LPS), and iNOSand IL-1β mRNA expression were quantified by TaqMan RT-PCR as a measureof the inflammatory response. Pretreatment with flavopiridolsubstantially reduced the inflammatory response. Importantly,flavopiridol treatment 3 hours after inflammatory stimulus was alsoeffective, indicating that a treatment window of ≥3 hours exists.

DETAILED DESCRIPTION

1. Introduction

The present invention is based, in part, on the discovery thatintervening with CDK9 activity finds us to preserve cartilage afterinjury (e.g., traumatic injury and/or surgical injury) and duringstorage (e.g., allograft storage). Any inhibitor of CDK9 known in theart can be used in the present methods, including inhibitory nucleicacids and inhibitory compounds (e.g., flavopiridol, and analogs andsalts thereof).

Many different stimuli can induce inflammation, and several of these arebeing investigated individually as arthritis drugs (e.g., IL-1antagonists, TNF antagonists, anti-oxidants). The focus has been oninhibition of the pathway so that transcription of response genes doesnot occur. None of these existing investigations have addressed theprocess of transcription. The present invention is based, in part, onthe discovery that all of these pathways converge on the activation ofCDK9 for the transcriptional elongation of the primary response genes.Inhibition of the transcriptional elongation by CDK9 is limited to theprimary response inflammatory genes, and CDK9 inhibition does not affecttranscription of housekeeping genes, and therefore is not detrimental tocells or tissues in the short term. The advantage of CDK9 inhibition isthat it reduces transcriptional elongation of inflammatory genes fromall inflammatory stimuli. In various embodiments, CDK9 can bespecifically and reversibly inhibited, e.g., with small-molecule drugs(e.g., flavopiridol and other known CDK9 inhibitors, and analogs andsalts thereof) and inhibitory nucleic acids (e.g., siRNA, miRNA,antisense RNA).

Symptomatic osteoarthritis (OA) can be defined as the end-stage failureof load bearing joints at the organ level (1). While the etiology of OAremains incompletely understood, it is well established that jointinjuries often progress to OA over time (2). High-energy joint traumasthat cause intra-articular fractures often result in the rapiddevelopment of joint degradation and post-traumatic osteoarthritis(PTOA) (3). Even lower-energy traumas to the joint, which are much morecommon, will initiate slowly progressing cartilage and joint degradationthat results in symptomatic PTOA many years later (2). As an example,from a total of 900,000 knee injuries annually in the United States (4),the American Academy of Orthopaedic Surgeons estimates 200,000 injuriesof the anterior cruciate ligament (ACL) in the general population (5),including 2500 to 3000 ACL reconstructions in military patients (6).Strikingly, approximately 50% of these ACL injury patients will developknee PTOA after a 10- to 20-year asymptomatic lag phase (7). NFLretirees under the age of 50 are five times more likely to havearthritis than comparable men in the general population, approximately80% of retirees report having joint pain lasting most of the day, andover 23% of NFL retirees over 50 years of age have had a jointreplacement.

Despite a lack of joint pain during the asymptomatic lag phase,progressive deterioration of bone and cartilage begins to develop soonafter traumatic joint injury. In OA of the knee or hip joints, theasymptomatic cartilage degeneration phase can last many years or evendecades (8). By the time arthritic joints become painful, there is oftenwidespread cartilage damage with areas of complete cartilage loss. As aresult of the extended painless “pre-OA” condition, the typical OApatient is seen in the clinic only after extensive joint damage hasalready occurred. At these late stages, treatment of the underlyingcauses of joint degeneration is no longer possible and the damage hasbecome irreversible. Current OA treatments address the associated jointpain, but do not improve joint function or alter the underlyingpathology. When these palliative treatments eventually fail, invasivesurgical joint replacement is the only remaining treatment for pain-freeambulation. Although there is abundant evidence that joint traumas suchas anterior cruciate ligament (ACL) tears will ultimately lead to OA(9-11), current clinical treatment does nothing at the time of theseinjuries to prevent the future onset of OA. Currently, clinicaltreatment is aimed at reducing the immediate pain and swelling in thejoint and restoring normal joint movement. The most commonrecommendations are to apply ice, gently compress the joint with anelastic bandage, and take pain medications such as aspirin,acetaminophen, or ibuprofen. Importantly, these treatments do notaddress the initiation of OA. The incidence of OA is independent ofwhether patients undergo surgical reconstruction of the ACL (7,12),suggesting that the injury event, in addition to the chronic jointinstability, has a causative role in OA pathogenesis.

The mechanical damage that a joint experiences during an impact hasimmediate effects on the tissues: cell death and physical damage to thejoint tissues occur within milliseconds of impact. The immediatemechanical damage then triggers an acute cellular response, which occurswithin a time-scale of minutes to hours (13). The acute response phaseis characterized by the release of inflammatory mediators from theinjured joint tissues, including IL-1, IL-6, iNOS, and TNF-α (13,14).This causes the transcriptional activation of primary response genes (orinflammatory genes), and leads to increased production of matrixdegrading enzymes such as MMPs, collagenases, aggrecanases, andcathepsins. The enzymatic degradation of matrix contributes to OA via acascade of destructive events, including:

-   -   (1) reducing the stiffness and elasticity of cartilage, thus        increasing the mechanical stresses on chondrocytes,    -   (2) increasing the hydraulic permeability of cartilage, leading        to loss of interstitial fluid and increased diffusion of solutes        (i.e. degradative enzymes, proteoglycans),    -   (3) increasing the accessibility of remaining cartilage matrix        structures to enzymatic digestion,    -   (4) thickening of the subchondral bone plate,    -   (5) structural changes to the trabecular bone, and    -   (6) formation of osteophytes and heterotopic bone (8).

We believe that a window for therapeutic intervention exists shortlyafter injury, during which attenuating the acute cellular responsedecreases production of matrix degrading enzymes and thus decreases thelikelihood of developing post-traumatic osteoarthritis (PTOA).

The transcriptional activation of primary response genes is an importantstep of the acute cellular response to injury. Transcriptionalactivation of primary response genes occurs in a timeframe of minutes tohours after the injury event. The majority of the primary response genesare ‘primed’ for transcription at a moment's notice, with thetranscription complex already assembled on the promoters and the RNApolymerase complex stalled just before entering the transcriptionelongation stage. In a recent Cell paper, Hargreaves et al elegantlydemonstrated that the rate-limiting step in transcriptional elongationof primary response genes is the recruitment of cyclin-dependentkinase-9 (CDK9) (15). In the case of inflammatory gene transcription,CDK9 is recruited to the transcription complex by NFκB (16,17).Importantly, CDK9 kinase activity is required for transcription of theprimary response inflammatory genes to proceed, and this mechanism ofregulation is conserved amongst primary response genes (18). Thus, CDK9kinase activity represents a new molecular target to inhibit the acuteinflammatory response after joint injury.

2. Subjects Who May Benefit

Subjects who can benefit from a regime of CDK9 inhibitors generally haveexperienced or imminently will experience an injury to cartilage tissue.For example, the subject may have experienced an injury (e.g., atraumatic injury) that damages cartilage tissue. The subject may alsoundergo or have undergone surgery to repair damaged cartilage tissueand/or to receive an osteochondral explant.

In various embodiments, a regime of a CDK9 inhibitor is administered tothe subject within about 10 days after damage or injury to cartilagetissue, for example, within about 9, 8, 7, 6, 5, 4, 3, 2 or 1 days afterdamage or injury to cartilage tissue. In various embodiments, a regimeof a CDK9 inhibitor is administered to the subject within about 24 hoursafter damage or injury to cartilage tissue, for example, within about22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hours afterdamage or injury to cartilage tissue.

3. Inhibitors of CDK9

Generally, the activity of a CDK9, e.g., a polypeptide having at least80% sequence identity, e.g., at least about 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an amino acidsequence of NP_001252.1, is inhibited or reduced, thereby preventing,reducing, delaying or inhibiting degradation of cartilage and/or onsetor progression of post-traumatic osteoarthritis.

a. Small Organic Compounds

CDK9 is a member of the cyclin-dependent kinase family, and mostproteins in this family regulate cell-cycle progression. Over the last 2decades there has been intense research into CDK inhibitors asanti-proliferative agents that arrest cell cycle progression in cancers,and numerous CDK inhibitors are in phase II and III clinical trials(19,20). CDK9, unlike most CDK proteins that regulate cell cycleprogression, is mainly thought to regulate RNA synthesis andtranscriptional elongation (21). There are small-molecule inhibitorswith relatively good specificity for CDK9, including flavopiridol, andanalogs and salts thereof. Commercial preparations of flavopiridol arecalled Alvocidib The IUPAC name for flavopiridol is2-(2-chlorophenyl)-5, 7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone). The structure offlavopiridol is shown below.

Flavopiridol inhibits CDK9 kinase activity by a high affinity (Kd=3 nMto 6 nM) interaction with the ATP-binding pocket of CDK9 (22,23).Inhibition of CDK9 kinase activity prevents transcriptional activationof primary response genes by preventing transcriptional elongation (15).Systemic administration of flavopiridol is well tolerated, and clinicaltrials with flavopiridol are successful in treating refractory chroniclymphocytic leukemia (24-26). Recently, Sekine et al have takenadvantage of the anti-proliferative effects of flavopiridol in mousemodels of rheumatoid arthritis (RA). They demonstrated that flavopiridolreduced synovial hyperplasia and effectively prevented rheumatoidarthritis (27). The anti-arthritic effect was reversible; whenflavopiridol treatment was stopped, synovial hyperplasia resumed and RAprogressed rapidly.

Other CDK inhibitors that can find use include without limitation, e.g.,4-(3,5-Diamino-1H-pyrazol-4-ylazo)-phenol (Calbiochem Catalog No.238811),2-(Pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridinone,PHA-767491 (Calbiochem Catalog No. 217707), and those described, e.g.,in International Publication Nos. WO 2012/101066 (pyridine biaryl aminecompounds); WO 2012/101065 (pyrimidine biaryl amine compounds); WO2012/101064 (N-acyl pyrimidine biaryl compounds); WO 2012/101063 (N-acylpyridine biaryl compounds); WO 2012/066070 (3-(aminoaryl)-pyridinecompounds); WO 2012/066065 (phenyl-heteroaryl amine compounds); WO2011/012661 (pyridine and pyrazine derivatives); WO 2011/077171(4-phenylamino-pyrimidine derivatives); WO 2010/020675(pyrrolopyrimidine compounds); WO 2008/079933 (heteroaryl-heteroarylcompounds); WO 2007/117653 (CDK9-PI3K-AKT inhibitors); WO 2006/024858(4-arylazo-3,5-diamino-pyrazole compounds); WO 2006/021803 (purine andpyrimidine CDK inhibitors) and in U.S. Patent Publication Nos.2012/0225899; 2012/0196855; 2012/0142680; 2010/0160350; 2010/0249149;2010/0076000; 2010/0035870; 2010/0003246; 2009/0325983; 2009/0318446;2009/0318441; 2009/0270427; 2009/0258886; 2009/0215805; 2009/0215805;2009/0137572; 2008/0125404; 2007/0275963; 2007/0225270; 2007/0072882;2007/0021452; 2007/0021419; and 2006/0264628, all of which are herebyincorporated herein by reference in their entirety for all purposes.

b. Inhibitory Nucleic Acids

Decreasing or inhibiting CDK9 gene expression can be achieved using anymethod in the art, including through the use of inhibitory nucleic acids(e.g., small interfering RNA (siRNA), micro RNA (miRNA), antisense RNA,ribozymes, etc.). Inhibitory nucleic acids can be single-strandednucleic acids that can specifically bind to a complementary nucleic acidsequence. By binding to the appropriate target sequence, an RNA-RNA, aDNA-DNA, or an RNA-DNA duplex or triplex is formed. Such inhibitorynucleic acids can be in either the “sense” or “antisense” orientation.See, for example, Tafech, et al., Curr Med Chem (2006) 13:863-81;Mahato, et al., Expert Opin Drug Deliv (2005) 2:3-28; Scanlon, CurrPharm Biotechnol (2004) 5:415-20; and Scherer and Rossi, Nat Biotechnol(2003) 21:1457-65.

In one embodiment, the inhibitory nucleic acid can specifically bind toa target nucleic acid sequence or subsequence that encodes a CDK9.Administration of such inhibitory nucleic acids can decrease or inhibitthe activity of CDK9 and consequently, cartilage degradation. Nucleotidesequences encoding CDK9 are known for several mammalian species,including human, e.g., NM_001261.3. From known CDK9 nucleotidesequences, one can derive a suitable inhibitory nucleic acid.

1. Antisense Oligonucleotides

In some embodiments, the inhibitory nucleic acid is an antisensemolecule. Antisense oligonucleotides are relatively short nucleic acidsthat are complementary (or antisense) to the coding strand (sensestrand) of the mRNA encoding a fungal specific CDK9. Although antisenseoligonucleotides are typically RNA based, they can also be DNA based.Additionally, antisense oligonucleotides are often modified to increasetheir stability.

Without being bound by theory, the binding of these relatively shortoligonucleotides to the mRNA is believed to induce stretches of doublestranded RNA that trigger degradation of the messages by endogenousRNAses. Additionally, sometimes the oligonucleotides are specificallydesigned to bind near the promoter of the message, and under thesecircumstances, the antisense oligonucleotides may additionally interferewith translation of the message. Regardless of the specific mechanism bywhich antisense oligonucleotides function, their administration to acell or tissue allows the degradation of the mRNA encoding CDK9.Accordingly, antisense oligonucleotides decrease the expression and/oractivity of CDK9.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. WO 89/10134), hybridization-triggered cleavageagents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) orintercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). Tothis end, the oligonucleotide can be conjugated to another molecule.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetyl cytosine, 5-(carboxyhydroxytriethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomet-hyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methyl cytosine, 5-methyl cytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also contain a neutral peptide-likebackbone. Such molecules are termed peptide nucleic acid (PNA)-oligomersand are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl.Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature 365:566.One advantage of PNA oligomers is their capability to bind tocomplementary DNA essentially independently from the ionic strength ofthe medium due to the neutral backbone of the DNA. In yet anotherembodiment, the antisense oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide isan—anomeric oligonucleotide. An anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

The selection of an appropriate oligonucleotide can be readily performedby one of skill in the art. Given the nucleic acid sequence encoding afungal-specific CDK9, one of skill in the art can design antisenseoligonucleotides that bind to a target nucleic acid sequence and testthese oligonucleotides in an in vitro or in vivo system to confirm thatthey bind to and mediate the degradation of the mRNA encoding the fungalspecific CDK9. To design an antisense oligonucleotide that specificallybinds to and mediates the degradation of a fungal-specific CDK9 encodingnucleic acid, it is preferred that the sequence recognized by theoligonucleotide is unique or substantially unique to the fungal specificCDK9 to be inhibited. For example, sequences that are frequentlyrepeated across an encoding sequence may not be an ideal choice for thedesign of an oligonucleotide that specifically recognizes and degrades aparticular message. One of skill in the art can design anoligonucleotide, and compare the sequence of that oligonucleotide tonucleic acid sequences that are deposited in publicly availabledatabases to confirm that the sequence is specific or substantiallyspecific for a fungal specific CDK9.

A number of methods have been developed for delivering antisense DNA orRNA to cells; e.g., antisense molecules can be injected directly intothe tissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systematically.

However, it may be difficult to achieve intracellular concentrations ofthe antisense sufficient to suppress translation on endogenous mRNAs incertain instances. Therefore another approach utilizes a recombinant DNAconstruct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. For example, a vectorcan be introduced in vivo such that it is taken up by a cell and directsthe transcription of an antisense RNA. Such a vector can remain episomalor become chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct that can be introduced directly into the tissue site.Alternatively, viral vectors can be used which selectively infect thedesired tissue, in which case administration may be accomplished byanother route (e.g., systematically).

2. Small Interfering RNA (siRNA or RNAi)

In some embodiments, the inhibitory nucleic acid is a small interferingRNA (siRNA or RNAi) molecule. RNAi constructs comprise double strandedRNA that can specifically block expression of a target gene. “RNAinterference” or “RNAi” is a term initially applied to a phenomenonwhere double-stranded RNA (dsRNA) blocks gene expression in a specificand post-transcriptional manner. RNAi provides a useful method ofinhibiting gene expression in vitro or in vivo. RNAi constructs caninclude small interfering RNAs (siRNAs), hairpin RNAs, and other RNAspecies which can be cleaved in vivo to form siRNAs. RNAi constructsherein also include expression vectors (“RNAi expression vectors”)capable of giving rise to transcripts which form dsRNAs or hairpin RNAsin cells, and/or transcripts which can produce siRNAs in vivo.

RNAi expression vectors express (transcribe) RNA which produces siRNAmoieties in the cell in which the construct is expressed. Such vectorsinclude a transcriptional unit comprising an assembly of (1) geneticelement(s) having a regulatory role in gene expression, for example,promoters, operators, or enhancers, operatively linked to (2) a “coding”sequence which is transcribed to produce a double-stranded RNA (two RNAmoieties that anneal in the cell to form an siRNA, or a single hairpinRNA which can be processed to an siRNA), and (3) appropriatetranscription initiation and termination sequences. The choice ofpromoter and other regulatory elements generally varies according to theintended host cell.

The RNAi constructs contain a nucleotide sequence that hybridizes underphysiologic conditions of the cell to the nucleotide sequence of atleast a portion of the mRNA transcript for the gene to be inhibited(i.e., a fungal-specific CDK9-encoding nucleic acid sequence). Thedouble-stranded RNA need only be sufficiently similar to natural RNAthat it has the ability to mediate RNAi. Thus, the invention has theadvantage of being able to tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism or evolutionarydivergence. The number of tolerated nucleotide mismatches between thetarget sequence and the RNAi construct sequence is no more than 1 in 5basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50basepairs. Mismatches in the center of the siRNA duplex are mostcritical and may essentially abolish cleavage of the target RNA. Incontrast, nucleotides at the 3′ end of the siRNA strand that iscomplementary to the target RNA do not significantly contribute tospecificity of the target recognition.

Sequence identity can be optimized by sequence comparison and alignmentalgorithms known in the art (see Gribskov and Devereux, SequenceAnalysis Primer, Stockton Press, 1991, and references cited therein) andcalculating the percent difference between the nucleotide sequences by,for example, the Smith-Waterman algorithm as implemented in the BESTFITsoftware program using default parameters (e.g., University of WisconsinGenetic Computing Group). Greater than 90% sequence identity, forexample, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity,between the inhibitory RNA and the portion of the target gene ispreferred. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mMPIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours;followed by washing).

Production of RNAi constructs can be carried out by chemical syntheticmethods or by recombinant nucleic acid techniques. Endogenous RNApolymerase of the treated cell may mediate transcription in vivo, orcloned RNA polymerase can be used for transcription in vitro. The RNAiconstructs may include modifications to either the phosphate-sugarbackbone or the nucleoside, e.g., to reduce susceptibility to cellularnucleases, improve bioavailability, improve formulation characteristics,and/or change other pharmacokinetic properties. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of an nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general response to dsRNA. Likewise, bases may be modified toblock the activity of adenosine deaminase. The RNAi construct may beproduced enzymatically or by partial/total organic synthesis, anymodified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis.

Methods of chemically modifying RNA molecules can be adapted formodifying RNAi constructs (see, for example, Heidenreich et al. (1997)Nucleic Acids Res, 25:776-780; Wilson et al. (1994) J Mol Recog 7:89-98;Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al.(1997) Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate,the backbone of an RNAi construct can be modified withphosphorothioates, phosphoramidate, phosphodithioates, chimericmethylphosphonate-phosphodie-sters, peptide nucleic acids,5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g.,2′-substituted ribonucleosides, a-configuration).

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition, while lower doses may also be useful for specificapplications. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition.

In certain embodiments, the subject RNAi constructs are “smallinterfering RNAs” or “siRNAs.” These nucleic acids are around 19-30nucleotides in length, and even more preferably 21-23 nucleotides inlength, e.g., corresponding in length to the fragments generated bynuclease “dicing” of longer double-stranded RNAs. The siRNAs areunderstood to recruit nuclease complexes and guide the complexes to thetarget mRNA by pairing to the specific sequences. As a result, thetarget mRNA is degraded by the nucleases in the protein complex. In aparticular embodiment, the 21-23 nucleotides siRNA molecules comprise a3′ hydroxyl group.

The siRNA molecules of the present invention can be obtained using anumber of techniques known to those of skill in the art. For example,the siRNA can be chemically synthesized or recombinantly produced usingmethods known in the art. For example, short sense and antisense RNAoligomers can be synthesized and annealed to form double-stranded RNAstructures with 2-nucleotide overhangs at each end (Caplen, et al.(2001) Proc Natl Acad Sci USA, 98:9742-9747; Elbashir, et al. (2001)EMBO J, 20:6877-88). These double-stranded siRNA structures can then bedirectly introduced to cells, either by passive uptake or a deliverysystem of choice, such as described below.

In certain embodiments, the siRNA constructs can be generated byprocessing of longer double-stranded RNAs, for example, in the presenceof the enzyme dicer. In one embodiment, the Drosophila in vitro systemis used. In this embodiment, dsRNA is combined with a soluble extractderived from Drosophila embryo, thereby producing a combination. Thecombination is maintained under conditions in which the dsRNA isprocessed to RNA molecules of about 21 to about 23 nucleotides.

The siRNA molecules can be purified using a number of techniques knownto those of skill in the art. For example, gel electrophoresis can beused to purify siRNAs. Alternatively, non-denaturing methods, such asnon-denaturing column chromatography, can be used to purify the siRNA.In addition, chromatography (e.g., size exclusion chromatography),glycerol gradient centrifugation, affinity purification with antibodycan be used to purify siRNAs.

In certain preferred embodiments, at least one strand of the siRNAmolecules has a 3′ overhang from about 1 to about 6 nucleotides inlength, though may be from 2 to 4 nucleotides in length. Morepreferably, the 3′ overhangs are 1-3 nucleotides in length. In certainembodiments, one strand having a 3′ overhang and the other strand beingblunt-ended or also having an overhang. The length of the overhangs maybe the same or different for each strand. In order to further enhancethe stability of the siRNA, the 3′ overhangs can be stabilized againstdegradation. In one embodiment, the RNA is stabilized by includingpurine nucleotides, such as adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine nucleotide 3′ overhangs by2′-deoxythyinidine is tolerated and does not affect the efficiency ofRNAi. The absence of a 2′ hydroxyl significantly enhances the nucleaseresistance of the overhang in tissue culture medium and may bebeneficial in vivo.

In other embodiments, the RNAi construct is in the form of a longdouble-stranded RNA. In certain embodiments, the RNAi construct is atleast 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, theRNAi construct is 400-800 bases in length. The double-stranded RNAs aredigested intracellularly, e.g., to produce siRNA sequences in the cell.However, use of long double-stranded RNAs in vivo is not alwayspractical, presumably because of deleterious effects which may be causedby the sequence-independent dsRNA response. In such embodiments, the useof local delivery systems and/or agents which reduce the effects ofinterferon are preferred.

In certain embodiments, the RNAi construct is in the form of a hairpinstructure (named as hairpin RNA). The hairpin RNAs can be synthesizedexogenously or can be formed by transcribing from RNA polymerase IIIpromoters in vivo. Examples of making and using such hairpin RNAs forgene silencing in mammalian cells are described in, for example,Paddison et al., Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature,2002, 418:38-9; McManus et al., RNA, 2002, 8:842-50; Yu et al., ProcNatl Acad Sci USA, 2002, 99:6047-52). Preferably, such hairpin RNAs areengineered in cells or in an animal to ensure continuous and stablesuppression of a desired gene. It is known in the art that siRNAs can beproduced by processing a hairpin RNA in the cell.

In yet other embodiments, a plasmid is used to deliver thedouble-stranded RNA, e.g., as a transcriptional product. In suchembodiments, the plasmid is designed to include a “coding sequence” foreach of the sense and antisense strands of the RNAi construct. Thecoding sequences can be the same sequence, e.g., flanked by invertedpromoters, or can be two separate sequences each under transcriptionalcontrol of separate promoters. After the coding sequence is transcribed,the complementary RNA transcripts base-pair to form the double-strandedRNA.

PCT application WO 01/77350 describes an exemplary vector forbi-directional transcription of a transgene to yield both sense andantisense RNA transcripts of the same transgene in a eukaryotic cell.Accordingly, in certain embodiments, the present invention provides arecombinant vector having the following unique characteristics: itcomprises a viral replicon having two overlapping transcription unitsarranged in an opposing orientation and flanking a transgene for an RNAiconstruct of interest, wherein the two overlapping transcription unitsyield both sense and antisense RNA transcripts from the same transgenefragment in a host cell.

RNAi constructs can comprise either long stretches of double strandedRNA identical or substantially identical to the target nucleic acidsequence or short stretches of double stranded RNA identical tosubstantially identical to only a region of the target nucleic acidsequence. Exemplary methods of making and delivering either long orshort RNAi constructs can be found, for example, in WO 01/68836 and WO01/75164.

Exemplary RNAi constructs that specifically recognize a particular gene,or a particular family of genes can be selected using methodologyoutlined in detail above with respect to the selection of antisenseoligonucleotide. Similarly, methods of delivery RNAi constructs includethe methods for delivery antisense oligonucleotides outlined in detailabove.

3. Ribozymes

In some embodiments, the inhibitory nucleic acid is a ribozyme.Ribozymes molecules designed to catalytically cleave an mRNA transcriptscan also be used to prevent translation of mRNA (See, e.g., PCTInternational Publication WO 90/11364; Sarver et al., 1990, Science247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleavemRNA at site-specific recognition sequences can be used to destroyparticular mRNAs, the use of hammerhead ribozymes is preferred.Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA have the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully inHaseloff and Gerlach, 1988, Nature, 334:585-591.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; WO 88/04300; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site that hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes that targeteight base-pair active site sequences.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) and canbe delivered to cells in vitro or in vivo. A preferred method ofdelivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme todestroy targeted messages and inhibit translation. Because ribozymesunlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

DNA enzymes incorporate some of the mechanistic features of bothantisense and ribozyme technologies. DNA enzymes are designed so thatthey recognize a particular target nucleic acid sequence, much like anantisense oligonucleotide, however much like a ribozyme they arecatalytic and specifically cleave the target nucleic acid.

There are currently two basic types of DNA enzymes, and both of thesewere identified by Santoro and Joyce (see, for example, U.S. Pat. No.6,110,462). The 10-23 DNA enzyme comprises a loop structure whichconnect two arms. The two arms provide specificity by recognizing theparticular target nucleic acid sequence while the loop structureprovides catalytic function under physiological conditions.

Briefly, to design an ideal DNA enzyme that specifically recognizes andcleaves a target nucleic acid, one of skill in the art must firstidentify the unique target sequence. This can be done using the sameapproach as outlined for antisense oligonucleotides. Preferably, theunique or substantially sequence is a G/C rich of approximately 18 to 22nucleotides. High G/C content helps insure a stronger interactionbetween the DNA enzyme and the target sequence.

When synthesizing the DNA enzyme, the specific antisense recognitionsequence that will target the enzyme to the message is divided so thatit comprises the two arms of the DNA enzyme, and the DNA enzyme loop isplaced between the two specific arms.

Methods of making and administering DNA enzymes can be found, forexample, in U.S. Pat. No. 6,110,462. Similarly, methods of delivery DNAribozymes in vitro or in vivo include methods of delivery RNA ribozyme,as outlined in detail above. Additionally, one of skill in the art willrecognize that, like antisense oligonucleotide, DNA enzymes can beoptionally modified to improve stability and improve resistance todegradation.

4. Formulation and Administration

In therapeutic applications, the CDK9 inhibitors can be administered toan individual who has suffered a traumatic injury to cartilage tissue,who has undergone surgery to repair cartilage tissue and/or who hasreceived a cartilage allograft. Compositions that contain CDK9inhibitors are administered to a patient in an amount sufficient tosuppress the undesirable inflammation and to eliminate or at leastpartially arrest symptoms and/or complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend on, e.g., the inhibitorcomposition, the manner of administration, the stage and severity of thedisease being treated, the weight and general state of health of thepatient, and the judgment of the prescribing physician. Inhibitors ofCDK9 activity can be administered chronically or acutely to reduce,inhibit or prevent cartilage degradation and post traumaticosteoarthritis. In certain instances, it will be appropriate toadminister an inhibitor of CDK9 activity prophylactically, for instancein subjects at risk of or suspected of developing cartilage degradationand/or post traumatic osteoarthritis.

Alternatively, DNA or RNA that inhibits expression of one or moresequences encoding a CDK9 protein, such as an antisense nucleic acid, asmall-interfering nucleic acid (i.e., siRNA), a micro RNA (miRNA), or anucleic acid that encodes a peptide that blocks expression or activityof a CDK9 can be introduced into patients to achieve inhibition. U.S.Pat. No. 5,580,859 describes the use of injection of naked nucleic acidsinto cells to obtain expression of the genes which the nucleic acidsencode.

Therapeutically effective amounts of CDK9 inhibitor or enhancercompositions of the present invention generally range for the initialadministration (that is for therapeutic or prophylactic administration)from about 0.1 μg to about 10 mg of CDK9 inhibitor for a 70 kg patient,usually from about 1.0 μg to about 1 mg, for example, between about 10μg to about 0.1 mg (100 μg). Typically, lower doses are initiallyadministered and incrementally increased until a desired efficaciousdose is reached. These doses can be followed by repeated administrationsover weeks to months depending upon the patient's response and conditionby evaluating symptoms associated with cartilage degradation and/orpost-traumatic osteoarthritis.

For prophylactic use, administration should be given to subjects at riskfor or suspected of developing cartilage degradation and/orpost-traumatic osteoarthritis. Therapeutic administration may beginconcurrently with surgical and/or allograft procedures, and/or as soonas possible after traumatic injury or surgery. This is often followed byrepeated administration until at least symptoms are substantially abatedand for a period thereafter. In some embodiments, the inhibitor of CDK9is administered within 10 days, e.g., within 9, 8, 7, 6, 5, 4, 3, 2, 1days, e.g., within 24, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1hours after experiencing traumatic injury. In some embodiments, thesubject has undergone surgery to repair damaged cartilage tissue. Insome embodiments, the subject has received an osteochondral explant,e.g., a cartilage allograft. In some embodiments, the inhibitor of CDK9is administered concurrently with or prior to surgery. In someembodiments, the inhibitor of CDK9 is administered within 10 days, e.g.,within 9, 8, 7, 6, 5, 4, 3, 2, 1 days, e.g., within 24, 20, 18, 16, 14,12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours after surgery. In someembodiments, the inhibitor of CDK9 is administered over a course of 10days, e.g., over 9, 8, 7, 6, 5, 4, 3, 2, 1 days. In various embodiments,the inhibitor of CDK9 is administered every 2 days, every day, or twicedaily, as appropriate.

The CDK9 inhibitors for therapeutic or prophylactic treatment areintended for systemic (e.g., parenteral, topical, oral, transdermal) orlocal (e.g., intralesional) administration. Preferably, the compositionsare formulated for oral administration. In certain embodiments, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, intranasally, inhalationally, subcutaneously,intradermally, or intramuscularly. Compositions of the invention arealso suitable for oral administration. Thus, the invention providescompositions for parenteral administration which comprise a solution ofthe CDK9 inhibiting agent dissolved or suspended in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carriersmay be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine oranother suitable amino acid, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

In embodiments where the CDK9 inhibitor is a small organic compound, thecompound (e.g., flavopiridol) and/or an analog thereof can beadministered orally, parenterally, (intravenously (IV), intramuscularly(IM), depo-IM, subcutaneously (SQ), and depo-SQ), sublingually,intranasally (inhalation), intrathecally, transdermally (e.g., viatransdermal patch), topically, ionophoretically or rectally. Typicallythe dosage form is selected to facilitate delivery to the brain (e.g.,passage through the blood brain barrier). In this context it is notedthat the compounds described herein are readily delivered to the brain.Dosage forms known to those of skill in the art are suitable fordelivery of the compound.

In varying embodiments, the CDK9 inhibitor is administeredintravenously. In embodiments where the CDK9 inhibitor is flavopiridol,dosing can be in accordance with concentrations and scheduling reportedin the art. For example, in various embodiments, flavopiridol isadministered intravenously in a concentration range of about 10 to about105 mg/m² in infusions delivered over 1 to 4 hours, as appropriate. See,e.g., Ramaswamy, et al., Invest New Drugs. (2012) 30(2):629-38; Phelps,et al., Blood. (2009) 113(12):2637-45; and Byrd, et al., Blood. (2007)109(2):399-404.

Compositions are provided that contain therapeutically effective amountsof the compound. The compounds are preferably formulated into suitablepharmaceutical preparations such as tablets, capsules, or elixirs fororal administration or in sterile solutions or suspensions forparenteral administration. Typically the compounds described above areformulated into pharmaceutical compositions using techniques andprocedures well known in the art.

These active agents (e.g., flavopiridol and/or analogs thereof) can beadministered in the “native” form or, if desired, in the form of salts,esters, amides, prodrugs, derivatives, and the like, provided the salt,ester, amide, prodrug or derivative is suitable pharmacologicallyeffective, e.g., effective in the present method(s). Salts, esters,amides, prodrugs and other derivatives of the active agents can beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry and described, for example, by March (1992)Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed.N.Y. Wiley-Interscience.

Methods of formulating such derivatives are known to those of skill inthe art. For example, the disulfide salts of a number of delivery agentsare described in PCT Publication WO 2000/059863 which is incorporatedherein by reference. Similarly, acid salts of therapeutic peptides,peptoids, or other mimetics, and can be prepared from the free baseusing conventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include, but are not limited to bothorganic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, orotic acid, and the like, aswell as inorganic acids, e.g., hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. An acidaddition salt can be reconverted to the free base by treatment with asuitable base. Certain particularly preferred acid addition salts of theactive agents herein include halide salts, such as may be prepared usinghydrochloric or hydrobromic acids. Conversely, preparation of basicsalts of the active agents of this invention are prepared in a similarmanner using a pharmaceutically acceptable base such as sodiumhydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide,trimethylamine, or the like. In certain embodiments basic salts includealkali metal salts, e.g., the sodium salt, and copper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH lower than the pKa of thedrug. Similarly, for the preparation of salt forms of acidic drugs, thepKa of the counterion is preferably at least about 2 pH higher than thepKa of the drug. This permits the counterion to bring the solution's pHto a level lower than the pHmax to reach the salt plateau, at which thesolubility of salt prevails over the solubility of free acid or base.The generalized rule of difference in pKa units of the ionizable groupin the active pharmaceutical ingredient (API) and in the acid or base ismeant to make the proton transfer energetically favorable. When the pKaof the API and counterion are not significantly different, a solidcomplex may form but may rapidly disproportionate (e.g., break down intothe individual entities of drug and counterion) in an aqueousenvironment.

Preferably, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

In various embodiments preparation of esters typically involvesfunctionalization of hydroxyl and/or carboxyl groups that are presentwithin the molecular structure of the active agent. In certainembodiments, the esters are typically acyl-substituted derivatives offree alcohol groups, e.g., moieties that are derived from carboxylicacids of the formula RCOOH where R is alky, and preferably is loweralkyl. Esters can be reconverted to the free acids, if desired, by usingconventional hydrogenolysis or hydrolysis procedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

The concentration of CDK9 inhibiting agents of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

The CDK9 inhibitors of the invention may also be administered vialiposomes, which can be designed to target the conjugates to aparticular tissue, for example, cartilage tissue. Liposomes includeemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. In thesepreparations, the peptide, nucleic acid or organic compound to bedelivered is incorporated as part of a liposome, alone or in conjunctionwith a molecule which binds to, e.g., a receptor prevalent among thedesired cells, or with other therapeutic compositions. Thus, liposomesfilled with a desired peptide, nucleic acid, small molecule or conjugateof the invention can be directed to the damaged or injured lesion, forexample, cartilage tissue, joints, injured lesions, where the liposomesthen deliver the selected CDK9 inhibitor compositions. Liposomes for usein the invention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, e.g., liposome size, acid liability and stabilityof the liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and4,837,028.

The targeting of liposomes using a variety of targeting agents is wellknown in the art (see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044).For targeting to desired cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the target cells. A liposome suspensioncontaining a CDK9 inhibitor may be administered intravenously, locally(e.g., intralesionally), topically, etc., in a dose which variesaccording to, inter alia, the manner of administration, the conjugatebeing delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more conjugates of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, the inhibitors are preferably supplied in asuitable form along with a surfactant and propellant. Typicalpercentages of CDK9 inhibitors are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included, asdesired, as with, e.g., lecithin for intranasal delivery.

An effective treatment is indicated by a decrease in observed symptoms(e.g., pain, swelling, joint mobility) as measured according to aclinician or reported by the patient. Alternatively, methods fordetecting levels of specific CDK9 activities can be used. Standardassays for detecting CDK9 activity are described herein. Again, aneffective treatment is indicated by a substantial reduction in activityof CDK9. As used herein, a “substantial reduction” in CDK9 activityrefers to a reduction of at least about 30% in the test sample comparedto an untreated control. Preferably, the reduction is at least about50%, more preferably at least about 75%, and most preferably CDK9activity levels are reduced by at least about 90% in a sample from atreated mammal compared to an untreated control. In some embodiments,the CDK9 activity is completely inhibited.

5. Matrices Comprising an Inhibitor of CDK9

In various embodiments, the CDK9 inhibitors can be contained within amatrix or a depot. The matrix can serve, in one capacity, as a deliveryvehicle for the composition to be delivered to the site of a cartilagelesion or a bone lesion. The matrix also provides a suitable scaffoldupon which cartilage repair and regeneration can occur. In oneembodiment, the matrix is bioresorbable or biodegradable.

In various embodiments, the matrix can be formed of any material that issuitable for in vivo use, and which provides the characteristicsfacilitating cartilage repair or bone repair in the presence of aninhibitor of CDK9. The matrix can be formed of materials which include,but are not limited to, synthetic polymers and/or a ground substance.Preferred ground substances include natural polymers and proteoglycans.Natural polymers include, but are not limited to collagen, elastin,reticulin and analogs thereof Proteoglycans include, but are not limitedto, any glycosaminoglycan-containing molecules. Particularly preferredglycosaminoglycans include chondroitin sulfate, dermatan sulphate,heparan sulphate, keratan sulphate and hyaluronan. Other preferredground substances include, but are not limited to, type I collagen, typeII collagen, type III collagen, type IV collagen and hyaluronic acid.Preferred synthetic polymers include poly(lactic acid) and poly(glycolicacid).

In one embodiment of the present invention, the matrix includescollagen. For example, the matrix can contain from about 20% to about100% collagen by dry weight of the matrix, for example, from about 50%to about 100% collagen by dry weight of the matrix, for example, fromabout 75% to about 100% collagen by dry weight of the matrix.

A matrix suitable for use with the inhibitors of CDK9 can includematerials in any suitable form for use in repairing a cartilage lesionor a bone lesion, including a sponge, a membrane, a film or a gel. Inone embodiment, a suitable repair matrix includes demineralized bonematrix, synthetic bone graft substitute, autograft tissue, allografttissue and/or xenograft tissue. In some embodiments, the matrix isformulated for use as a bone graft, for example, as a spinal graft.

Suitable methods for associating an inhibitor of CDK9 with a matrixinclude any method which allows the inhibitors to be delivered to a siteof cartilage repair or bone repair together with the matrix such thatthe cartilage repair or bone repair product is effective to repairand/or regenerate cartilage or bone at the site. Such methods ofassociation include, but are not limited to, suspension of thecomposition within the matrix, freeze-drying of the composition onto asurface of the matrix and suspension within the matrix of acarrier/delivery formulation containing the composition. Additionally,the inhibitors of CDK9 can be associated with the matrix prior toplacement of the product into a cartilage lesion (i.e., the associationof the composition with matrix occurs ex vivo) or alternatively, thematrix can first be implanted into a lesion, followed by association ofthe inhibitors of CDK9 with the matrix, such as by injection into or ontop of the matrix (i.e., the association of the composition with matrixoccurs in vivo).

The inhibitors of CDK9 can contain additional delivery formulations orcarriers which enhance the association of the composition with thematrix, which enhance the delivery of the composition to the appropriatecells and tissue at the site of the lesion, and which assist incontrolling the release of the factors in the composition at the site ofthe lesion. Suitable delivery formulations include carriers, which, asused herein, include compounds that increase the half-life of acartilage-inducing composition in the treated animal. Suitable carriersinclude, but are not limited to, polymeric controlled release vehicles,biodegradable implants, liposomes, bacteria, viruses, oils, cells,esters, and glycols. Preferably, the matrices are bioresorbable orbiodegradable.

The inhibitors of CDK9 are present in the matrix at a concentration thatis effective to induce, at the site of a cartilage lesion or a bonelesion, one or more of: cellular infiltration, cellular proliferation,angiogenesis, and cellular differentiation to type II collagen-producingchondrocytes. Preferably, the inhibitors of CDK9 are present in thematrices at a concentration that is effective to induce cartilage repairand/or regeneration at the site of a cartilage lesion or a bone lesion.One of skill in the art will be able to adjust the concentration ofproteins and/or nucleic acid molecules in the composition depending onthe types and number of proteins to be provided by the composition, andthe delivery vehicle used.

The matrices can also contain one or more substances that non-covalentlyattach to the inhibitors of CDK9 in the composition and thus, modify therelease rate of the growth factor. Such substances include, but are notlimited to, any ground substance or other polymeric substance. As usedherein, a ground substance is defined as the non-living matrix ofconnective tissue, which includes natural polymers and proteoglycans.Natural polymers include, but are not limited to collagen, elastin,reticulin and analogs thereof. Proteoglycans include, but are notlimited to any glycosaminoglycan-containing molecules, and includechondroitin sulfate, dermatan sulphate, heparan sulphate, keratansulphate and hyaluronan. Preferred ground substances include, but arenot limited to, type I collagen, type II collagen, type III collagen,type IV collagen and hyaluronic acid. Preferred other polymericsubstances include, but are not limited to, poly(lactic acid) andpoly(glycolic acid).

In a further embodiment, the matrices can include one or more types ofcells which are provided to further enhance chondrogenesis at the siteof the cartilage lesion. Such cells include, but are not limited to,fibrochondrocytes, chondrocytes, mesenchymal precursors, and any othercell that can serve as a chondrocyte precursor. Such cells can beassociated with the composition and the matrix by any of the methodsdescribed above.

In some aspects of the present invention, matrices comprising theinhibitors of CDK9 further comprise at least one bone matrix protein. Asused herein, “bone matrix proteins” are any of a group of proteins knownin the art to be a component of or associated with the minutecollagenous fibers and ground substances which form bone matrix. Invarious embodiments, the matrices comprise a bone matrix protein that isa member of the TGF-β superfamily, a growth factor protein and/orCartilage Oligomeric Matrix Protein (COMP). Bone matrix proteins canalso include, but are not limited to, osteocalcin, osteonectin, bonesialoprotein (BSP), lysyloxidase, cathepsin L pre, osteopontin, matrixGLA protein (MGP), biglycan, decorin, proteoglycan-chondroitin sulfateIII (PG-CS III), bone acidic glycoprotein (BAG-75), thrombospondin (TSP)and/or fibronectin. Preferably, bone matrix proteins suitable for usewith the product of the present invention include one or more of:osteocalcin, osteonectin, MGP, TSP, BSP, lysyloxidase and cathepsin Lpre. In one embodiment, the at least one bone matrix protein includes atleast osteocalcin, osteonectin, BSP, lysyloxidase and cathepsin L pre. Aparticularly preferred bone matrix protein is MGP, and more preferred isosteonectin, and most preferred is TSP.

The matrices comprising the inhibitors of CDK9 are useful for repairinga variety of defects in cartilage, including both tears and segmentaldefects in both vascular and avascular cartilage tissue. The product isparticularly useful for repairing defects in hyaline (e.g., articular)and/or fibrocartilage (e.g., meniscal). For example, matrices comprisinginhibitors of CDK9 find use promoting repair of a meniscal radial tear;a meniscal triple bucket handle tear; a longitudinal tear in theavascular area of a meniscus; or a meniscal segmental lesion.

Because cartilage defects and bone defects (i.e., lesions) can occur ina variety of shapes, sizes, and locations, a matrix comprising theinhibitors of CDK9 is of a shape and size sufficient to conform to aspecific defect in the cartilage or the bone of the patient to betreated. Preferably, the matrix, when used in the repair of a cartilagedefect or bone defect, achieves a geometry at the defect site that issuitable to provide a therapeutic benefit to the patient. Such atherapeutic benefit can be any improvement in a patient's health andwell-being that is related to a correction of the cartilage defect orthe bone defect, and preferably, the therapeutic benefit includes therepair of the defect such that the natural configuration of thecartilage or the bone is at least partially restored. The matrix can befixed or implanted directly into a cartilage lesion or a bone lesion.

6. Compositions and Kits

In a related aspect, the invention provides compositions comprising anosteochondral explant (e.g., ex vivo cartilage tissue) and/orchondrocytes in a solution comprising an inhibitor of cyclin-dependentkinase 9 (CDK9). In some embodiments, the osteochondral explant isallograft cartilage. In some embodiments, the inhibitor of CDK9 is asmall organic compound, as described above. In varying embodiments, theinhibitor of CDK9 is flavopiridol, or analogs and salts thereof. In someembodiments, the osteochondral explant is submerged in the solutioncomprising the inhibitor of CDK9. In varying embodiments, the solutionis an aqueous solution, e.g., a physiologically isotonic solution. Insome embodiments, the solution comprises flavopiridol at a concentrationin the range of about 100 nM to about 1000 nM, e.g., about 300 nM. Thesolution may contain additional pharmaceutically acceptable excipients,described herein. In some embodiments, the composition is provided as apackaged kit.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 CDK9 Inhibition Protects Cartilage from the Catabolic Effectsof Pro-Inflammatory Cytokines

Methods

Human Articular Chondrocytes—

Primary chondrocytes were isolated from cartilage tissues obtained fromtotal knee arthroplasty according to IRB approved protocol and culturedas monolayer (Li et al., Osteoarthritis Cartilage. (2011)19(10):1246-53). The chondrocytes were used for experiments within 3-5days without passaging to avoid dedifferentiation.

Treatments of Chondrocytes—

Primary chondrocytes grown in 6-well plates (˜80% confluence) weretreated with 10 ng/ml lipopolysaccharide (LPS) (Sigma), or 10 ng/mlIL-1β (R&D System) for 5 hours, with or without 300 nM CDK9 inhibitorflavopiridol (Sigma). The cells were washed 3 times with PBS andharvested for RNA extraction.

Quantitative Real-Time PCR—

Total RNA were extracted using the RNeasy Mini Kit (Qiagen) and reversetranscribed using a Superscript first-strand kit (Invitrogen). RT-PCRwas performed in triplicates using a 7900HT real-time PCR system(Applied Biosystem) with gene specific probes (Applied Biosystem) andnormalized to 18s rRNA, or alternatively, with the PCR Arrays for HumanNFκB Signaling Targets (Qiagen, cat. no. 330231), according to themanufacturers' protocol. PCR array data were analyzed by theaccompanying online analysis software provided by Qiagen on the internetat qiagen.com.

Assessment of Cartilage Degradation—

Human cartilage explants (about 3 mm cubes) were treated with 1 ng/mlIL-1b for 6 days, in the presence or absence of 6 or 300 nM Flavopiridol(with media change on day 3). The amount of glycosaminoglycan (GAG)released into the media was determined by the colorimetricdimethylmethylene blue dye-assay, with chondroitin sulfate as standard(Farndale, et al., Biochim Biophys Acta 883:173-177). The release ofCol2a degradation products into the media was determined by measuringthe amount of cleaved Col2a peptides (Poole, et al., J Immunol Methods(2004) 294:145-153.) with the C2C ELISA kit (IBEX Pharmaceuticals)according to the manufacturer's protocol.

Statistical Analysis—

Values of all measurements were expressed as the mean+standarddeviation. Statistical comparison was performed by two-tailed Student'st test. Values of p<0.05% were considered significant.

Results

Flavopiridol inhibits LPS-induced inflammatory response in chondrocytes.To test whether CDK9 inhibitor flavopiridol inhibits the transcriptionalactivation of primary inflammatory response genes, human articularchondrocytes were treated with LPS for 5 hours. As expected, thisactivated transcription of the primary response genes IL-1β and iNOS(FIG. 1). However, pre-treatment with 300 nM flavopiridol stronglyattenuated the transcriptional activation of both genes. Importantly,addition of flavopiridol 3 hours after LPS treatment still markedlyinhibited IL-1β and iNOS activation (FIG. 1).

Flavopiridol Prevents IL-1β-Induced Expression of Matrix-DegradingEnzymes in Chondrocytes.

IL-1β induces a host of MMPs and ADAMTSs that degrade collagen andaggrecan, respectively, in cartilage matrix. We tested whether CDK9inhibition could effectively suppress this in chondrocytes. Upon IL-1βstimulation, the expression of MMPs and ADAMTSs were activated asexpected. However, co-treatment with flavopiridol effectively suppressedthe activation of these genes (FIG. 2) (except cathepsin B (CTSB), whichwas not activated by IL-1β). These data indicate that CDK9 activity isimportant for the activation of cartilage-degrading enzymes followinginflammatory stimulation.

Flavopiridol suppresses a broad range of primary inflammatory responsegenes. We next tested the effectiveness of flavopiridol in inhibitingthe IL-1β-mediated activation of multiple primary inflammatory responsegenes in chondrocytes, using a NF-κB target PCR array (SABiosciences).As shown in FIG. 3, IL-1β strongly activated many NF-κB-dependentprimary response genes (presented as fold-induction over untreatedcontrol on a logarithmic scale, in the absence (blue bars) or presence(red bars) of flavopiridol. The percent inhibition by flavopiridol wasshown on top of each bars). In most cases, flavopiridol effectivelyabolished the activation (>90%) of these genes. For example, IL-1βactivated IL-6 by 492-fold, but only 4.1-fold in the presence offlavopiridol. Importantly, housekeeping genes are largely unaffected byeither IL-1β or flavopiridol.

CDK9 controls the activation of inflammatory response from diversesignals. Although the rate-limiting step for transcriptional activationof inflammatory response genes in lymphocytes is controlled by CDK9(Hargreaves et al., Cell. (2009) 138(1):129-45; Zippo, et al., Cell(2009) 138:1122-36), its role in regulating the innate inflammatoryresponse in articular chondrocytes has not been investigated. To thisend, we tested the involvement of CDK9 in the activation of inflammatoryresponse genes in chondrocytes simulated by three different inflammatorysignals; namely, Interleukin 1 beta (IL-1β), Lipopolysaccharides (LPS),and Tissue Necrosis Factor alpha (TNFα). Cellular response to IL-1β,LPS, or TNFα is mediated by three distinct pathways—activation of theIL-1Receptor, Toll-Like Receptor 4, or TNF Receptor 1, respectively(FIG. 4A). Chondrocytes were treated with the above three agentsindependently, in the presence or absence of the CDK9 inhibitorFlavopiridol. The mRNA expression of the inducible nitric oxide synthase(iNOS) (Maier et al., Biochim Biophys Acta. (1994) 1208(1):145-50), acommon effector gene for all three pathways, was then determined toassess the immune response in chondrocytes. The results showed thatFlavopiridol greatly suppressed the activation of iNOS expression in allthree pathways (FIG. 4B), demonstrating the effectiveness andversatility of Flavopiridol in preventing inflammatory response fromdiverse signals. Thus our data confirmed previous finding in othersystems and established CDK9 as a central regulatory point for innateinflammatory response in chondrocytes.

CDK9 inhibition prevents the activation of a broad spectrum ofinflammatory response genes. To further investigate the effects of CDK9inhibition on the activation of other inflammatory mediators besidesiNOS, the gene expression profiles of chondrocytes treated with IL-1βfor 5 hrs were determined by real-time PCR arrays. Each PCR arraycontained 84 key genes responsive to NFκB signal transduction (Qiagen),which regulates multiple cellular processes such as inflammatory,immunity, and stress responses. The average gene expression profilesfrom three chondrocyte donors were presented as heat maps, in which lowand high relative expressions were represented by green and red colors,respectively (FIG. 5). The results showed that IL-1β strongly activatedthe majority of these NFκB-target genes (FIG. 5, compared lane 1& 2),while CDK9 inhibition by Flavopiridol almost completely abolished theeffects of IL-1β (lane 3). On average, across three chondrocyte donors,CDK9 inhibition repressed IL-1β activity by >86%, with respect to 54 outof 60 NFκB-target genes (listed in FIG. 5) that were activated by atleast 2-fold under out experimental conditions. Importantly,housekeeping genes were not affected by either IL-1β or Flavopiridol.These data demonstrated that CDK9 can be targeted to effectivelysuppress the activation of a cascade of downstream inflammatory responsegenes.

CDK9 inhibition prevents the activation of catabolic genes inchondrocytes. Besides activating the acute phase inflammatory genes,pro-inflammatory cytokines such as IL-1β and TNFα can also stimulate theexpression of catabolic genes in chondrocytes (Goldring, et al., AnnRheum Dis (2008) 67 Suppl 3:iii75-82; Kobayashi, et al., Arthritis Rheum(2005) 52:128-135). These catabolic genes include the various MMPs andADAMTS4 (aggrecanase) that degrade the cartilage matrix. Given the roleof CDK9 in activation of inflammatory genes, we next examined theeffects of CDK9 inhibition in the induction of MMPs and ADAMTS4 inchondrocytes treated with IL-1β. The results showed that IL-1β-mediatedup-regulation of MMP1, 3, 9, and 13, as well as ADAMTS4 mRNAs wasmarkedly suppressed by co-treatment with Flavopiridol. These dataindicated that CDK9 inhibition prevents the activation of catabolicgenes in chondrocytes.

CDK9 inhibition protects cartilage from the catabolic effects of IL-1β.Since CDK9 inhibition suppresses activation of inflammatory andcatabolic genes in chondrocytes, we next determined whether Flavopiridolcan protect cartilage from the deleterious effects of pro-inflammatorycytokines. To this end, cartilage explants were isolated and cultured inmedia containing 1 ng/mL IL-1β, in the presence or absence ofFlavopiridol for 6 days. Degradation of cartilage matrix was assessed bymeasuring the release of GAG and Col2a cleavage peptides into theculturing media. As expected, IL-1β alone increased the amount of bothGAG (FIG. 7) and Col2a peptides (FIG. 8) released into the media(compared first and second bars). However, the concentrations of bothGAG and Col2a peptides were reduced by 6 nM Flavopiridol and returned tobaseline levels by 300 nM Flavopiridol (FIGS. 7 and 8). Thus our dataprovided evidence that CDK9 inhibition prevented the catabolicdestruction of cartilage by IL-1β.

Discussion

The etiology of primary OA remains incompletely understood and theinvolvement of inflammation is controversy. However, it iswell-established that damage to Col2a originates around chondrocytes atthe cartilage matrix surface (Hollander, et al., J Clin Invest (1995)96:2859-2869.). Since inflammatory response induces chondrocyteapoptosis and cartilage matrix breakdown (Goldring, et al., Ann RheumDis (2008) 67 Suppl 3:iii75-82), there are several anti-OA strategiesthat target either specific branches of the inflammatory signalingcascade (e.g. IL-1, IL-6, TNFα, and NFκB inhibitors) (Kobayashi, et al.,Arthritis Rheum (2005) 52:128-135; Attur, et al., OsteoarthritisCartilage (2011) 19:1158-1164; Attur, et al., J Biol Chem (2000)275:40307-40315), or the downstream events such as apoptosis withcaspase inhibitors (Lotz, et al., Arthritis Res Ther. (2007) 12:211).However, because inflammation can be induced by a variety of stimuli,the above individual approaches would have limited effectiveness inhandling the diverse challenges in a biological system, as well aslimited abilities in efficiently suppressing a broad range ofinflammatory mediator expression. Our novel and unique approach tosolving this problem is to directly target CDK9 that activatestranscription of primary inflammatory response genes. Using thepharmacological CDK9 inhibitor Flavopiridol, we have shown cartilage canbe protected from the harmful effects of pro-inflammatory cytokines.

Our results demonstrate for the first time in chondrocytes thatFlavopiridol effectively suppress the innate immune response to multipleinflammatory stimuli (FIG. 4), and prevented the induction of a host ofinflammatory mediators (FIG. 5), as well as MMPs and aggrecanase thatdegrade the cartilage matrix (FIG. 6). In most cases, Flavopiridolalmost completely abolishes the activation of inflammatory mediatorexpression. For example, from the PCR array data (FIG. 2), IL-1β inducedexpression of IL-6 by 492-fold, but only 4.2-fold in the presence ofFlavopiridol, representing a 99.2% repression of IL-1β-dependentinflammatory response gene transcription. These data are furthersupported by our observation that less IL-1β-induced matrix degradationproducts of GAG and Col2a is detected in cartilage explants treated withFlavopiridol (FIGS. 7 and 8). The reduction in matrix degradationproducts is not due to the loss of cell viability in cartilage treatedwith Flavopiridol, because live/dead staining revealed similarchondrocyte viabilities between control and Flavopiridol treatedcartilage.

Flavopiridol is an ATP analog that selectively inhibits CDK9 kinaseactivity by a high affinity interaction with its ATP-binding pocket (Ni,et al., PLoS One. (2010) 5(11):e13792). Flavopiridol was originallyknown for its anti-proliferation properties by suppressing cell-cycleprogression. Its pharmacological activity is well-documented over thelast two decades because of its use in clinical trials asanti-proliferation/cancer agent (reviewed by Wang and Ren, Mini Rev MedChem. (2010) 10(11):1058-70). Taken advantage of the anti-proliferativeeffects of Flavopiridol, Sekine et al have demonstrated that systemicadministration of Flavopiridol reduced synovial hyperplasia andprevented rheumatoid arthritis (RA) in a collagen-induced mouse model(Sekine et al., J Immunol. (2008) 180(3):1954-61). However, we believedthe anti-RA activity of Flavopiridol is likely due to the systematicsuppression of B-cell-mediated immune response to the injected collagen,rather than the localized suppression of the innate immune response incartilage. Our group has developed a non-invasive post-traumatic OA(PTOA) model in mouse (Christiansen et al., Osteoarthritis Cartilage.(2012) 20(7):773-82) useful for testing the ability of Flavopiridol andother CDK9 inhibitors to prevent OA, optionally in conjunction withother existing PTOA models.

In summary, our data for the first time demonstrate the absoluterequirement of CDK9 activity in the activation of primary inflammatoryresponse genes in human chondrocytes. In addition, our results stronglyindicate that Flavopiridol is an effective and versatile agent toprevent activation of acute inflammatory response and catabolic pathwaysin cartilage. The present data show the effectiveness of flavopiridol insuppressing an inflammatory response in chondrocytes, and thus itstherapeutic implications in preventing cartilage breakdown in jointinjuries, or in osteochondral explants. CDK9 inhibitors thus provide anew strategy to prevent or delay the onset of OA.

Example 2 CDK9 Inhibition Prevents or Delays the Onset of Long-TermPost-Traumatic Osteoarthritis (PTOA)

Our model of mouse knee injury consistently causes PTOA within 8 weeks.Injured and uninjured mice will be treated with either CDK9 inhibitor orsaline control. Treatment with CDK9 inhibitor will be initiated earlyafter joint injury, and maintained for the duration of maximal primaryresponse gene activation. The treatment duration should not exceed 7days post-injury. (A) the long-term development of PTOA, (B) the levelof serum OA biomarkers, and (C) the changes in subchondral bonemicrostructure by micro-computed tomography (μCT) are measured.

Currently accepted mouse models of PTOA rely on non-physiologicalmethods to induce OA, including using a needle to induce cruciatetransection in a ‘closed knee’, damaging knee ligaments using ‘openknee’ surgical techniques (Glasson, et al., Nature, (2005) 434(7033):644-8; Kamekura, et al., Osteoarthritis and cartilage/OARS,Osteoarthritis Research Society, (2005) 13(7): 632-41; Glasson, et al.,Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society,(2007) 15(9): 1061-9), applying repeated bouts of supraphysiologicalmechanical loads (Poulet, et al., Arthritis & Rheumatism, (2011) 63(1):137-147), and directly injecting collagenase (van Osch, et al., TheJournal of Rheumatology, (1996) 23(7): 1227-32; Joosten, et al., Am JPathol, (2004) 165(3): 959-67) or chemical agents such as iodoacetate(Ameye, et al., Curr Opin Rheumatol, (2006) 18(5): 537-47) into thejoint space. While these existing models all initiate OA, they areinvasive and non-physiological and do not faithfully mimic the commonlow-energy human knee traumas such as ACL tears. In addition, theinvasive nature of the surgical procedures is likely to initiate aninflammatory response of its own, obfuscating studies of the naturalcourse of the inflammatory response associated with an ACL tear.

We have developed a whole-joint injury model to initiate post-traumaticOA in mice, in which a single rapid non-invasive mechanical load inducesACL rupture. This model faithfully replicates a clinically relevanthuman knee injury, enabling us to focus on the natural early events ofjoint injury that initiate the subsequent progression of OA. Our modelrepresents an advance over other animal models of PTOA, since it is anoninvasive injury that mimics a clinically relevant situation. Theinjuries are highly reproducible and easy to perform, which enables usto design experiments with more variables and obtain statisticallysignificant results using fewer animals. As with other more invasivePTOA mouse models (Glasson, et al., Nature, (2005) 434(7033): 644-8), OAdevelops consistently within 8-12 weeks of injury. As in human kneeinjuries, we observe an initial acute inflammatory response and jointswelling that resolves in a few days, and extensive remodeling ofsubchondral bone and cartilage. Also comparable to human knee injuries(Dahlberg, et al., Ann Rheum Dis, (1994) 53: 823-7), we observe asystemic inflammatory response that results in similar (although lowermagnitude) structural changes in the contralateral uninjured knee.

To generate knee injuries, the mouse is anesthetized using isofluraneinhalation, and then the right leg of each mouse is subjected to tibialcompression loading, as shown in FIG. 10. The tibial compression systemconsists of two custom-designed loading platens. The bottom platen holdsthe flexed knee, and the top platen holds the foot with the ankleslightly flexed. The platens are aligned vertically and positionedwithin an electromagnetic materials testing machine (Bose EnduraTecElectroForce 3200, Eden Prairie, Minn.). A single axial compressiveload, at a loading rate of 1 millimeter per second, is applied to atarget compressive load of 12 Newtons (N) to induce the knee injury.

In this model, joint injury reproducibly occurs at a compressivemagnitude between 10N to 11N and is evident by an abrupt change in theforce profile, and a visible dislocation of the knee joint.Osteoarthritis consistently develops within 8 weeks. The typicalhistology of an injured joint and contralateral uninjured joint 8-weekspost-injury is shown in FIG. 11. A significant loss of proteoglycans isobserved by 8 weeks, as well as hypertrophy and calcification of themeniscus. Postmortem analysis using high-resolution x-ray microscopy(XRadia, VersaXRM-500, Pleasanton Calif.) and standard micro-computedtomography (SCANCO, μCT 35, Bassersdorf Switzerland) indicated that theACL was disrupted in all mice examined, typically due to a tibial ACLavulsion fracture. Osteophytes and heterotopic bone formation werepresent by 8-weeks post-injury, predominantly on the medial andposterior aspects of the joint. Quantitative analysis of bone volume inthe μCT images indicated that the injury induced a pronounced initialloss of trabecular bone volume that was evident within 3 dayspost-injury, shown in FIG. 12. There was maximal bone loss 7 days afterinjury (40% and 44% loss of trabecular bone volume at the tibial andfemoral epiphysis, respectively). The bone volume was slowly regained byweek 4, although not to its original value (˜80% of the value on day 1).The observation of such rapid remodeling of trabecular bone is novel,and further emphasizes the need for early intervention after jointinjury. Analysis of the contralateral uninjured knee revealed similarchanges in bone volume, though to a much lesser extent. The observationof a systemic response to the localized injury supports clinical studieswhich found elevated markers of joint degradation in the uninjured kneeof patients with ACL rupture injuries (Dahlberg, et al., Ann Rheum Dis,(1994) 53: 823-7).

In summary, we have developed a non-invasive joint injury model in micethat closely mimics clinically relevant human joint injuries. This newinjury model is an improvement to existing models, which usenon-physiological or invasive methods to create injury, methods thatcould obfuscate studies of the primary response gene activation.

Our mouse model of joint injury consistently causes PTOA within 8 weeks,and this is in agreement with the many other models of mouse PTOA.Delaying the onset of PTOA to 3 or 4 months in mice would represent asubstantial improvement in disease progression. Injured and uninjuredmice are treated with either CDK9 inhibitor or saline control. Initialtreatment is within 1 to 24 hours of injury, and that the treatmentduration does exceed 7 days post-injury. We measure (A) the long-termdevelopment of PTOA, (B) serum biomarkers of OA, and (C) changes insubchondral bone microstructure by μCT.

Treatment Groups:

Mice are injured as described above, or undergo anesthesia and handlingbut without receiving joint injury. Injured and uninjured mice will betreated with either CDK9 inhibitor or saline control. Mice are randomlyassigned to one of four groups: injured or uninjured, and treated withflavopiridol or vehicle control. Treated mice receive ckd9 inhibitorflavopiridol administered systemically through an IP injection asdescribed by Sekine et al (Sekine, et al., Journal of Immunology, (2008)180(3): 1954-61), and control mice receive vehicle only (0.01% DMSO insaline). Mice are injured as described above, or undergo anesthesia andhandling but without receiving joint injury.

Histological Assessment of PTOA.

Histology is the standard method to evaluate OA in mice knees. To assessthe progression of PTOA in a semi-quantitative manner, the guidelinesset forth by the OARSI histopathology initiative late last year arefollowed (Glasson, et al., Osteoarthritis and cartilage/OARS,Osteoarthritis Research Society, (2010) 18 Suppl 3: S17-23, 2010;Aigner, et al., Osteoarthritis and cartilage/OARS, OsteoarthritisResearch Society, 18 Suppl 3: S2-6). Briefly, mouse knees will bedissected to remove skin and excess muscle and paraffin-embedded.Frontal sections, serially harvested at 80-100 μm increments, areobtained to cover the entire articulating surface. Sections are stainedfor proteoglycan content using Safranin-O and counterstained with FastGreen. At least 2 trained individuals perform the grading of OA usingthe recommended scoring system (Glasson, et al., Osteoarthritis andcartilage/OARS, Osteoarthritis Research Society, (2010) 18 Suppl 3:S17-23). Evaluators are without any knowledge of the treatment or injurystatus of the sections. OA scores are compared in across the treatmentgroups using the JMP 9.0 statistical package.

Serum Biomarkers of OA.

Serum biomarkers are promising for monitoring the clinical progressionof OA. Most of the serum biomarkers rely on quantifying the breakdownproducts of matrix components specific to articular cartilage. A fewrely on the increased synthesis of collagen-specific proteins, forexample the pro-domain of type II collagen that is shed during collagenfibrillogenesis. One of the few serum biomarkers also validated in mouseserum is cartilage oligomeric matrix protein (COMP). The presence ofCOMP is quantified in the mouse serum, using commercially available kits(MD BioProducts, Zurich Switzerland). Serum levels of COMP will becompared across groups and with time, in combination with the OA scoresfrom above, to determine the efficacy of CDK9 inhibition in preventingPTOA.

MicroCT Analysis.

Remodeling of subchondral bone, an advancing tidemark with increasedcalcification of the articular cartilage and meniscus, and osteophyteformation are all hallmarks of osteoarthritis that can be quantifiedusing microCT analysis. Mice will be scanned 1 day before injury, andthen again at the indicated time points. An in-vivo micro-computedtomography instrument (SCANCO vivaCT-40, Bassersdorf, Switzerland) willbe used, and various aspects of bone microstructure quantified using theincluded software. Comparisons between groups and across time will beperformed to determine the efficacy of CDK9 inhibition in preventingPTOA.

Timecourse:

This example focuses on the prevention or delay of PTOA in the longterm. OA consistently develops within 2 months in our model. A delay inthe onset of OA by 2 additional months is considered an advance in thetreatment of mouse PTOA, and holds promise for translation into theclinical setting. PTOA is evaluated at 2, 3, and 4 months post-injury.

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of reducing or inhibiting cartilagedegradation and/or chondrocyte death in a subject who has experiencedtraumatic joint injury, comprising administering to the subject aneffective amount of a small organic compound inhibitor ofcyclin-dependent kinase 9 (CDK9) or salt thereof, wherein the traumaticjoint injury triggers an acute cellular response characterized by therelease of inflammatory mediators from the injured joint tissues,wherein the inhibitor of CDK9 or salt thereof is initially administeredwithin 4 days after experiencing the traumatic joint injury, therebyreducing or inhibiting cartilage degradation and/or chondrocyte death inthe subject.
 2. The method of claim 1, wherein the subject hasexperienced a traumatic injury to cartilage tissue.
 3. The method ofclaim 2, wherein the subject has undergone joint surgery.
 4. The methodof claim 3, wherein the inhibitor of CDK9 is administered concurrentlywith or prior to surgery.
 5. The method of claim 3, wherein theinhibitor of CDK9 is administered within 24 hours after surgery.
 6. Themethod of claim 1, wherein the inhibitor of CDK9 is initiallyadministered within 24 hours after experiencing traumatic joint injury.7. The method of claim 1, wherein the subject has undergone surgery torepair damaged cartilage tissue.
 8. The method of claim 1, wherein thesubject has received an osteochondral explant.
 9. The method of claim 8,wherein the osteochondral explant is a cartilage allograft.
 10. Themethod of claim 1, wherein the inhibitor of CDK9 is administeredsystemically.
 11. The method of claim 10, wherein the inhibitor of CDK9is administered intravenously.
 12. The method of claim 1, wherein theinhibitor of CDK9 is administered directly to the site of injuredcartilage tissue.
 13. The method of claim 12, wherein the inhibitor ofCDK9 is delivered from a matrix.
 14. The method of claim 1, wherein theinhibitor of CDK9 specifically or preferentially inhibits CDK9 kinaseactivity.
 15. The method of claim 1, wherein the inhibitor of CDK9inhibits CDK9 kinase activity with a Kd of less than about 6 nM.
 16. Themethod of claim 1, wherein the inhibitor of CDK9 inhibits CDK9 kinaseactivity by interaction with the ATP-binding pocket of CDK9.
 17. Themethod of claim 1, wherein the inhibitor of CDK9 is a biaryl compoundselected from the group consisting of a pyridine biaryl amine compound;a pyrimidine biaryl amine compound; an N-acyl pyrimidine biarylcompound; an N-acyl pyridine biaryl compound; a 3-(aminoaryl)-pyridinecompound; and a phenyl-heteroaryl amine compound.
 18. A method ofreducing, delaying or inhibiting the onset and/or progression ofpost-traumatic osteoarthritis in a subject who has experienced atraumatic joint injury, comprising administering to the subject aneffective amount of a small organic compound inhibitor ofcyclin-dependent kinase 9 (CDK9) or salt thereof, wherein the traumaticjoint injury triggers an acute cellular response characterized by therelease of inflammatory mediators from the injured joint tissues,wherein the inhibitor of CDK9 or salt thereof is initially administeredduring the acute response phase and reduces and/or inhibitstranscriptional activation of the primary response genes after the jointinjury, thereby reducing or inhibiting post-traumatic osteoarthritis inthe subject.
 19. The method of claim 18, wherein the primary responsegenes comprise one or more of IL-10, inducible nitric oxide synthase(iNOS), IL-6, TNF-.alpha., MMP-1, MMP-3, MMP-9, MMP-13 and ADAMTS4(aggrecanase).
 20. The method of claim 18, wherein the inhibitor or CDK9or salt thereof is administered to the subject within about 10 daysafter damage or injury.
 21. The method of claim 18, wherein theinhibitor or CDK9 or salt thereof is initially administered within 4days after experiencing traumatic joint injury.
 22. The method of claim18, wherein the inhibitor or CDK9 or salt thereof is initiallyadministered within 3 days after experiencing traumatic injury.
 23. Themethod of claim 18, wherein the inhibitor or CDK9 or salt thereof isinitially administered within 2 days after experiencing traumaticinjury.
 24. The method of claim 18, wherein the inhibitor or CDK9 orsalt thereof is initially administered within 24 hours afterexperiencing traumatic joint injury.
 25. The method of claim 18, whereinthe inhibitor or CDK9 or salt thereof is initially administered within 3hours after experiencing traumatic joint injury.
 26. A method ofreducing or inhibiting degradation of an osteochondral explant and/orreducing or inhibiting chondrocyte death during storage, comprisingstoring the osteochondral explant and/or chondrocytes in a solutioncomprising an effective amount of a small organic compound inhibitor ofcyclin-dependent kinase 9 (CDK9) or salt thereof.
 27. A method ofreducing or inhibiting cartilage degradation and/or chondrocyte death ina subject who has experienced traumatic joint injury, comprisingadministering to the subject an effective amount of an inhibitor ofcyclin-dependent kinase 9 (CDK9) or salt thereof, wherein the CDK9inhibitor is selected from the group consisting of4-(3,5-Diamino-1H-pyrazol-4-ylazo)-phenol (Calbiochem Catalog No.238811), and2-(Pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridinone,PHA-767491 (Calbiochem Catalog No. 217707), wherein the traumatic jointinjury triggers an acute cellular response characterized by the releaseof inflammatory mediators from the injured joint tissues, wherein theinhibitor of CDK9 or salt thereof is initially administered within 4days after experiencing the traumatic joint injury, thereby reducing orinhibiting cartilage degradation and/or chondrocyte death in thesubject.
 28. The method of claim 1, wherein the subject is administeredrepeated administrations of the inhibitor of CDK9 or salt thereof. 29.The method of claim 1, wherein the inhibitor or CDK9 or salt thereof isadministered over a course of 10 days.
 30. The method of claim 1,wherein the inhibitor or CDK9 or salt thereof is initially administeredwithin 3 days after experiencing traumatic injury.
 31. The method ofclaim 1, wherein the inhibitor or CDK9 or salt thereof is initiallyadministered within 2 days after experiencing traumatic injury.
 32. Themethod of claim 1, wherein the inhibitor or CDK9 or salt thereof isinitially administered within 3 hours after experiencing traumatic jointinjury.